Metabolic engineering of Bacillus subtilis for growth on overflow metabolites

Abstract

BackgroundThe genome of the important industrial host Bacillus subtilis does not encode the glyoxylate shunt, which is necessary to utilize overflow metabolites, like acetate or acetoin, as carbon source. In this study, the operon encoding the isocitrate lyase (aceB) and malate synthase (aceA) from Bacillus licheniformis was transferred into the chromosome of B. subtilis. The resulting strain was examined in respect to growth characteristics and qualities as an expression host.ResultsOur results show that the modified B. subtilis strain is able to grow on the C2 compound acetate. A combined transcript, protein and metabolite analysis indicated a functional expression of the native glyoxylate shunt of B. lichenifomis in B. subtilis. This metabolically engineered strain revealed better growth behavior and an improved activity of an acetoin-controlled expression system.ConclusionsThe glyoxylate shunt of B. licheniformis can be functionally transferred to B. subtilis. This novel strain offers improved properties for industrial applications, such as growth on additional carbon sources and a greater robustness towards excess glucose feeding.

Metabolic engineering of Bacillus subtilis forl‐valine overproduction

“…glutamicum (Bott & Eikmanns, 2013). However, acetate metabolism was markedly improved in B. subtilis via the expression of isocitrate lyase and malate synthase from Bacillus licheniformis, effectively completing the glyoxylate shunt in B. subtilis (Kabisch et al, 2013).…”

“…While direct comparisons of experimental results obtained under significantly different conditions may not be entirely meaningful, our experiences with batch and fed‐batch cultivations indicate that acetate dissimilation is limiting l ‐valine overproduction in AW018‐5 (data not shown). The slow rate of acetate dissimilation can be attributed to the absence of a glyoxylate shunt in B. subtilis (Kabisch et al, ), which is present in both E. coli (Maloy & Nunn, ) and C. glutamicum (Bott & Eikmanns, ). However, acetate metabolism was markedly improved in B. subtilis via the expression of isocitrate lyase and malate synthase from Bacillus licheniformis , effectively completing the glyoxylate shunt in B. subtilis (Kabisch et al, ).…”

“…The slow rate of acetate dissimilation can be attributed to the absence of a glyoxylate shunt in B. subtilis (Kabisch et al, ), which is present in both E. coli (Maloy & Nunn, ) and C. glutamicum (Bott & Eikmanns, ). However, acetate metabolism was markedly improved in B. subtilis via the expression of isocitrate lyase and malate synthase from Bacillus licheniformis , effectively completing the glyoxylate shunt in B. subtilis (Kabisch et al, ). Moreover, strategies such as promoter engineering to modulate the expression of aceE , encoding the E1p subunit of the pyruvate dehydrogenase complex (Buchholz et al, ), and activating the glyoxylate shunt via guided evolution of isocitrate dehydrogenase in a phosphoenolpyruvate carboxylase‐ and pyruvate carboxylase‐deficient mutant (Schwentner et al, ), dramatically improved l ‐valine overproduction in C. glutamicum without acetate supplementation.…”

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli

“…However as amylases are expressed only under nutrient limitation, even with Bacilli an initial growth control is possible (own non-published experiments). A better control is obtained if amyE knockouts are used [ 87 ]. Ploss et al [ 88 ] used the slow glucose release of the growth system to mimic industrial carbon-limited growth conditions of B. subtilis to investigate the expression heterogeneity of secretory proteins with α-amylase AmyM from Geobacillus stearothermophilus as case example.…”

Evaluation of small organic acids present in fast pyrolysis bio‐oil from lignocellulose as feedstocks for bacterial bioconversion

“…While E. coli, P. putida, and C. glutamicum possess the key enzymes of the glyoxylate cycle and thus are able to grow on acetate as sole carbon source (Cortay et al, 1989;Gerstmeir et al, 2003;Kornberg & Krebs, 1957;Reinscheid, Eikmanns, & Sahm, 1994a;Sudarsan, Dethlefsen, Blank, Siemann-Herzberg, & Schmid, 2014;Wendisch et al, 2000), B. subtilis lacks enzymes required for the glyoxylate cycle (Freese & Fortnagel, 1969). To enable growth of B. subtilis on acetate, Kabisch et al (2013) transferred an operon encoding the two key enzymes of the glyoxylate cycle, ISL and MS, from B. licheniformis.…”